Method for the reduction of host cell proteins in affinity chromatography

ABSTRACT

The current invention reports a method for purifying an antibody by reducing the content of a host cell protein. The method employs a wash step with a low conductivity aqueous solution in an affinity chromatography.

The present invention relates to the field of purification ofpolypeptides. The present invention in particular relates to thereduction of host cell proteins like phospholipase B-like 2 (PLBL2) orClusterin in solutions containing antibodies.

BACKGROUND OF THE INVENTION

Proteins and especially immunoglobulins play an important role intoday's medical portfolio. For human application every therapeuticprotein has to meet distinct criteria. To ensure the safety ofbiopharmaceutical agents to humans by-products accumulating during theproduction process have to be removed especially. To fulfill theregulatory specifications one or more purification steps have to followthe manufacturing process. Among other things, purity, throughput, andyield play an important role in determining an appropriate purificationprocess.

Different methods are well established and widespread used for proteinpurification, such as affinity chromatography (e.g. protein A or proteinG affinity chromatography, single chain Fv ligand affinitychromatography), ion exchange chromatography (e.g. cation exchange(sulfopropyl or carboxymethyl resins), anion exchange (amino ethylresins) and mixed-mode ion exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis).

For the purification of recombinantly produced immunoglobulins often acombination of different column chromatography steps is employed. Duringthe purification non-immunoglobulin contaminants such as host cellprotein and host cell DNA as well as endotoxins and viruses aredepleted. Therefore, generally an affinity chromatography step, likeprotein A affinity chromatography is followed by one or more additionalseparation steps. In general, high conductivity buffers are described tobe employed in wash steps of affinity chromatrography methods.

In U.S. Pat. No. 6,127,526 a method for purifying proteins by Protein Achromatography is described which comprises the steps of: (a) adsorbingthe protein to Protein A immobilized on a solid phase comprising silicaor glass; (b) removing contaminants bound to the solid phase by washingthe solid phase with a hydrophobic electrolyte solvent; and (c)recovering the protein from the solid phase.

In WO2011/038894 a protein A chromatography method with a pronounceddepletion of host cell protein and DNA by specific wash steps prior tothe recovery of the immunoglobulin from the protein A chromatographicmaterial is reported.

In WO2013/177118 compositions and methods for the isolation andpurification of antibodies from a sample matrix are reported.

In WO2013/033517 methods for separating a polypeptide of interest (suchas an antibody) from a virus are reported.

A method for purifying a protein, including one or more chromatographicprocesses, in which an amino acid; or a dipeptide, an oligopeptide, or apolyamino acid thereof is included in a buffer solution used in at leastone chromatographic process (equilibration buffer, wash buffer, andelution buffer), thereby purifying a high-purity protein with a verysmall quantity of the impurity (e.g., polymers or host cell proteins) isreported in EP2583973.

SUMMARY OF THE INVENTION

Herein is reported a method for the production of an antibody withreduced content of host cell proteins by purifying the antibody with anaffinity chromatography step.

In more detail it has been found that by the method of the currentinvention which uses a low conductivity aqueous solution in a wash stepof an affinity chromatography prior to the recovery of an antibody fromthe chromatographic material, that the content of certain host cellproteins in a solution comprising the antibody can be reduced.Accordingly, it has been found that the content of phospholipases (inparticular phospholipase B-like 2 (PLBL2)) can be reduced. It has beenfound that the PLBL2 content can be reduced 100-fold or more if theantibody is of the IgG4 isotype.

One aspect as reported herein is the use of a low conductivity aqueoussolution in a wash step of a protein A chromatography for reducing thecontent of a host cell protein wherein the protein A chromatography isused to purify a human IgG1 or a human IgG4 isotype antibody.

In one embodiment of this aspect the human IgG4 isotype antibody is anantibody against P-selectin, or an bispecific antibody against factorIXa and factor X, or an antibody against IL-13, or an antibody againstamyloid beta. In one embodiment of this aspect the human IgG1 isotypeantibody is an antibody against Influenza B, or an antibody againstVEGF-A, or an antibody against CD22, or a bispecific antibody againstHER3 and EGFR, or an antibody against amyloid beta, or an antibodyagainst Her2, or a bispecific antibody against Ang2 and VEGF-A, or abispecific antibody against carcinoembryonic antigen (CEA) and CD3.

In one embodiment of this aspect the low conductivity aqueous solutionhas a conductivity value of about 0.5 mS/cm or less.

In one embodiment of this aspect the host cell protein is phospholipaseB-like 2 (PLBL2) or Clusterin.

In one embodiment of this aspect the low conductivity aqueous solutioncomprises about 0.1 mM to about 8 mM Tris.

In one embodiment of this aspect the low conductivity aqueous solutioncomprises about 0.05 mM to about 2 mM potassium phosphate.

In one embodiment of this aspect the low conductivity aqueous solutionhas a pH of about 7 or higher.

In one embodiment of this aspect the low conductivity aqueous solutionwash step is preceded or succeeded by a high conductivity aqueoussolution wash step.

In one embodiment of this aspect the high conductivity aqueous solutionhas a conductivity value of about 20 mS/cm or higher.

In one embodiment of this aspect an intermediate wash step is performedwith a medium conductivity aqueous solution between the low conductivityaqueous solution wash step and the high conductivity aqueous solutionwash step.

In one embodiment of this aspect the medium conductivity aqueoussolution has a conductivity value of from more than 0.5 mS/cm to lessthan 20 mS/cm.

In one embodiment of this aspect the high (or medium) conductivityaqueous solution comprises Histidine.

One aspect as reported herein is a method for producing a human IgG4 orIgG1 isotype antibody comprising the steps of

-   -   a) cultivating a cell comprising a nucleic acid encoding a human        IgG4 or IgG1 isotype antibody,    -   b) recovering the human IgG4 or IgG1 isotype antibody from the        cell or the cultivation medium,    -   c) contacting the human IgG4 or IgG1 isotype antibody with a        protein A chromatography material,    -   d) washing the protein A chromatography material with a low        conductivity aqueous solution,    -   e) recovering the human IgG4 or IgG1 isotype antibody from the        protein A chromatography material        and thereby producing the human IgG4 or IgG1 isotype antibody.

One aspect as reported herein is method for purifying a human IgG4 orIgG1 isotype antibody from a sample comprising the steps of

-   -   a) providing a sample comprising a human IgG4 or IgG1 isotype        antibody,    -   b) purifying the human IgG4 or IgG1 isotype antibody with a        protein A chromatography method/step, comprising washing the        protein A chromatography material with a low conductivity        aqueous solution.

In one embodiment of all aspects the human IgG4 isotype antibody is anantibody against P-selectin or a bispecific antibody against factor IXaand factor X or an antibody against IL-13 or an antibody against amyloidbeta. In one embodiment of all aspects the human IgG1 isotype antibodyis an antibody against Influenza B or an antibody against VEGF-A or anantibody against CD22 or a bispecific antibody against HER3 and EGFR oran antibody against amyloid beta or an antibody against Her2 or abispecific antibody against Ang2 and VEGF-A, or a bispecific antibodyagainst carcinoembryonic antigen (CEA) and CD3.

In one embodiment of all aspects the low conductivity aqueous solutionhas a conductivity value of about 0.5 mS/cm or less.

In one embodiment of all aspects the content of a host cell protein isreduced and the (specific) host cell protein is phospholipase B-like 2(PLBL2) or Clusterin.

In one embodiment of all aspects the low conductivity aqueous solutioncomprises about 0.1 mM to about 8 mM Tris.

In one embodiment of all aspects the low conductivity aqueous solutioncomprises about 0.05 mM to about 2 mM potassium phosphate.

In one embodiment of all aspects the low conductivity aqueous solutionhas a pH of about 7 or higher.

In one embodiment of all method aspects the method additionallycomprises washing the affinity chromatography material with a highconductivity aqueous solution and/or with a medium conductivity aqueoussolution before or after washing the protein A chromatography materialwith low conductivity aqueous solution.

In one embodiment of all aspects the high conductivity aqueous solutionhas a conductivity value of about 20 mS/cm or higher.

In one embodiment of all aspects the medium conductivity aqueoussolution has a conductivity value of from more than 0.5 mS/cm to lessthan 20 mS/cm.

In one embodiment of all aspects the high or medium conductivity aqueoussolution comprises Histidine.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported an improved affinity chromatography method and usecomprising the washing of the affinity chromatography material with alow conductivity aqueous solution.

It has been found that specific host cell proteins can be reduced with awash step with a low conductivity aqueous solution, when this wash stepis used in an affinity chromatography step, e.g. a protein Achromatrography step. The affinity chromatography step is used in apurification or production method for antibodies. The low conductivityaqueous solution wash step is particularly effective to reduce thecontent of phospholipase B-like 2 (PLBL2).

One aspect as reported herein is the use of a low conductivity aqueoussolution in a wash step of an affinity chromatography for reducing thecontent of a (specific) host cell protein.

One aspect as reported herein is a method for producing a human IgGisotype antibody comprising

-   -   a) cultivating a cell comprising a nucleic acid encoding the        human IgG isotype antibody,    -   b) recovering the human IgG isotype antibody from the cell or        the cultivation medium,    -   c) contacting (a solution comprising) the human IgG isotype        antibody with an affinity chromatography material,    -   d) washing the affinity chromatography material with a low        conductivity aqueous solution, while at least 90% the bispecific        antibody remains bound to the affinity chromatography material,    -   e) recovering the human IgG isotype antibody from affinity        chromatography material        and thereby producing the human IgG isotype antibody.

One aspect as reported herein is a method for purifying a human IgGisotype antibody from a sample comprising the steps of

-   -   a) providing a (buffered aqueous) sample comprising a human IgG        isotype antibody,    -   b) purifying the human IgG isotype antibody with a affinity        chromatography method/step, comprising washing the affinity        chromatography material with low conductivity aqueous solution.

Recombinant polypeptides produced in CHO cells may be purified accordingto the methods described herein to remove or reduce levels of a hostcell proteins.

Exemplary recombinant polypeptides include therapeutic antibodies andimmunoadhesins, including, without limitation, antibodies, includingantibody fragments, to one or more of the following antigens: HER1(EGFR), HER2 (e.g., trastuzumab, pertuzumab), HER3, HER4, VEGF (e.g.,bevacizumab, ranibizumab), MET (e.g., onartuzumab), CD20 (e.g.,rituximab, obinutuzumab, ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18,an ICAM, VLA-4, VCAM, IL-17A and/or F, IgE (e.g., omalizumab), DRS,CD40, Apo2L/TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta7(e.g., etrolizumab), IL-13 (e.g., lebrikizumab), Abeta (e.g.,crenezumab, gantenerumab), P-selectin (e.g., inclacumab), IL-6R (e.g.,tociluzumab), IFNa (e.g., rontalizumab), M1prime (e.g., quilizumab),mitogen activated protein kinase (MAPK), OX40L, TSLP, Factor D (e.g.,lampalizumab) and receptors such as: IL-9 receptor, IL-5 receptor,IL-4receptor alpha, IL-13receptoralphal and IL-13receptoralpha2, OX40,TSLP-R, IL-7R alpha (a co-receptor for TSLP), IL17RB (receptor forIL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, FcepsilonRI andFcepsilonRII/CD23 (receptors for IgE). Other exemplary antibodiesinclude those selected from, and without limitation, antiestrogenreceptor antibody, anti-progesterone receptor antibody, anti-p53antibody, anticathepsin D antibody, antiBcl-2 antibody, anti-E-cadherinantibody, anti-CA125 antibody, anti-CA15-3 antibody, antiCA19-9antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEAantibody, Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody,anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8antibody, anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11cantibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody,anti-CD19 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody,anti-CD38 antibody, anti-CD41 antibody, antiLCA/CD45 antibody,anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody,anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99 antibody,anti-CD106 antibody, antiubiquitin antibody, anti-CD71 antibody,anti-c-myc antibody, anti-cytokeratins antibody, antivimentins antibody,anti-HPV proteins antibody, anti-kappa light chains antibody,anti-lambda light chains antibody, anti-melanosomes antibody,anti-prostate specific antigen antibody, antiS-100 antibody, anti-tauantigen antibody, anti-fibrin antibody, anti-keratins antibody andantiTn-antigen antibody.

In some embodiments, exemplary antibodies include antibodies to Abeta,antibodies to IL17 A/F and antibodies to CMV. Exemplary anti-Abetaantibodies and methods of producing such antibodies have been describedpreviously, for example, in WO2008011348, WO2007068429, WO2001062801,and WO2004071408. Exemplary anti-IL17 A/F antibodies and methods ofproducing such antibodies have been described previously, for example,in WO 2009136286 and U.S. Pat. No. 8,715,669. Exemplary anti-CMVantibodies, including anti-CMV-MSL, and methods of producing suchantibodies have been described previously, for example, in WO2012047732.

In some embodiments the affinity chromatography is used to purify ahuman IgG isotype antibody. In some embodiments the affinitychromatography is used to purify an IgG4 antibody. In one embodiment theIgG4 isotype antibody is an antibody against P-selectin or a(bispecific) antibody against factor IXa and factor X or an antibodyagainst IL-13 or an antibody against amyloid beta. In some embodimentsthe affinity chromatography is used to purify an IgG1 isotype antibody.In one embodiment the IgG1 isotype antibody is an antibody againstInfluenza B or an antibody against VEGF-A or an antibody against CD22 oran (bispecific) antibody against HER3 and EGFR or an antibody againstamyloid beta or an antibody against Her2 or a bispecific antibodyagainst Ang2 and VEGF-A or a bispecific antibody againstcarcinoembryonic antigen (CEA) and CD3.

One aspect as reported herein is a method for producing a human IgG4isotype antibody (containing solution) comprising

-   -   a) cultivating a cell comprising a nucleic acid encoding a human        IgG4 isotype antibody,    -   b) recovering the human IgG4 isotype antibody from the cell or        the cultivation medium,    -   c) contacting the human IgG4 isotype antibody with an affinity        chromatography material,    -   d) washing the affinity chromatography material with a low        conductivity aqueous solution,    -   e) recovering the human IgG4 isotype antibody from the affinity        chromatography material        and thereby producing the human IgG4 isotype antibody.

One aspect as reported herein is a method for producing an IgG4 isotypeantibody (containing solution) comprising

-   -   a) cultivating a cell comprising a nucleic acid encoding an IgG4        isotype antibody,    -   b) recovering the IgG4 isotype antibody from the cell or the        cultivation medium,    -   c) contacting the IgG4 isotype antibody with an affinity        chromatography material,    -   d) washing the affinity chromatography material with a low        conductivity aqueous solution,    -   e) recovering the IgG4 isotype antibody from the affinity        chromatography material        and thereby producing the IgG4 isotype antibody.

One aspect as reported herein is a method for purifying a human IgG4isotype antibody from a sample comprising the steps of

-   -   a) providing a sample comprising a human IgG4 isotype antibody,    -   b) purifying the human IgG4 isotype antibody with a affinity        chromatography method/step, comprising washing the affinity        chromatography material with a low conductivity aqueous        solution.

One aspect as reported herein is a method for purifying an IgG4 isotypeantibody from a sample comprising the steps of

-   -   a) providing a sample comprising an IgG4 isotype antibody,    -   b) purifying the IgG4 isotype antibody with a affinity        chromatography method/step, comprising washing the affinity        chromatography material with a low conductivity aqueous        solution.

It has been found that the content of a host cell protein can be reducedif the conductivity of the aqueous solution used in the wash step is lowi.e a low conductivity aqueous solution is used for washing. In oneembodiment of all aspects the low conductivity aqueous solution has aconductivity value of about 1 mS/cm or less. In one preferred embodimentof all aspects the low conductivity aqueous solution has a conductivityvalue of about 0.5 mS/cm or less. In one embodiment the low conductivityaqueous solution has a conductivity value of from about 0.03 μS/cm toabout 0.5 mS/cm. In one embodiment the low conductivity aqueous solutionhas a conductivity value of from about 0.05 μS/cm to about 0.35 mS/cm.In one embodiment of all aspects the low conductivity aqueous solutionis deionized water. For some applications deionized water is notsuitable to be used in a wash step. In some embodiments the lowconductivity aqueous solution is not deionized water.

It has been found that a protein A affinity chromatography can be usedfor the purposes as reported herein. In one preferred embodiment of allaspects the affinity chromatography is a protein A affinitychromatography. In one embodiment the protein A affinity chromatographyis selected from the group comprising MabSelectSure affinitychromatography, ProSep vA affinity chromatography, Poros Mab Capture Aaffinity chromatography, ProSep Ultra Plus affinity chromatography,MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein A-650F;Toyopearl AF-rProtein A HC-650HF). In one embodiment the affinitychromatography is a protein G affinity chromatography. In one embodimentthe affinity chromatography is an affinity chromatography that uses arecombinant protein as a ligand, that means that the affinitychromatography is a recombinant protein ligand affinity chromatography.In one embodiment the affinity chromatography is an affinitychromatography that uses a single chain Fv as a ligand, that means thatthe affinity chromatography is a single chain Fv ligand affinitychromatography. In one embodiment the affinity chromatography comprisesa mutated Protein A coupled to a chromatography matrix or a fragment ofProtein A coupled to a chromatography matrix.

It has been found that the content of (specific) host cell proteins canbe reduced. It has been found that especially the content ofphospholipase B-like 2 (PLBL2) can be reduced. In one embodiment the(specific) host cell protein is a Chinese hamster ovary (CHO) host cellprotein. In one preferred embodiment of all aspects the (specific) hostcell protein is phospholipase B-like 2 (PLBL2) or Clusterin. In oneembodiment the (specific) host cell protein is phospholipase B-like 2(PLBL2).

It has been found that low conductivity aqueous solution may comprisecertain buffering substances e.g. Tris or potassium phosphate in lowamounts. In one embodiment the low conductivity aqueous solutioncontains tris(hydroxymethyl)aminomethane (Tris). In one embodiment thelow conductivity aqueous solution comprises about 0.1 mM to about 10 mMTris. In one embodiment the low conductivity aqueous solution comprisesabout 0.5 mM to about 6.5 mM Tris. In one embodiment the lowconductivity aqueous solution comprises about 2 mM Tris. In oneembodiment the low conductivity aqueous solution contains potassiumphosphate. In one embodiment the low conductivity aqueous solutioncomprises about 0.05 mM to about 5 mM potassium phosphate. In oneembodiment the low conductivity aqueous solution comprises about 0.05 mMto about 2 mM potassium phosphate. In one embodiment the lowconductivity aqueous solution comprises about 0.5 mM potassiumphosphate.

It has been found that the effect of reducing the content of a host cellprotein is pronounced if the low conductivity aqueous solution has acertain pH. In one embodiment the low conductivity aqueous solution hasa pH of about 7 or higher. In one embodiment the low conductivityaqueous solution has a pH of about 7.5 or higher. In one embodiment thelow conductivity aqueous solution has a pH of from about 7 to about 9.5.In one embodiment the low conductivity aqueous solution has a pH of fromabout 7.5 to about 8.5. In one embodiment the low conductivity aqueoussolution has a pH of about 8. In one embodiment the low conductivityaqueous solution has a pH of about 9.

It has been found that the effect of reducing the content of a host cellprotein can also be achieved if the pH of the low conductivity aqueoussolution is about 8.5 or higher and the low conductivity aqueoussolution has a conductivity value of about 1.2 mS/cm or less. In oneembodiment the low conductivity aqueous solution has a pH of about 8.5or higher and the low conductivity aqueous solution has a conductivityvalue of about 1.2 mS/cm or less. In one embodiment the low conductivityaqueous solution has a pH of about 8.5 or higher and the lowconductivity aqueous solution has a conductivity value of about 1 mS/cmor less. In one embodiment low conductivity aqueous solution has a pH ofabout 8.5 or higher and the low conductivity aqueous solution comprisesabout 55 mM Tris or less. In one embodiment low conductivity aqueoussolution has a pH of about 8.5 or higher and the low conductivityaqueous solution comprises about 30 mM Tris or less.

In one embodiment the low conductivity aqueous solution is in the pHrange of from pH 7 to less than pH 8.5 and has a conductivity value ofabout 0.5 mS/cm or less and at a pH value of 8.5 or more a conductivityvalue of about 1.2 mS/cm or less.

It has been found that by the uses and the methods as reported hereinthe content of host cell proteins like PLBL2 can be reduced to a certainlevel, e.g. when compared to the load amount of PLBL2 prior to apurification step like an affinity chromatography step. In oneembodiment the content of PLBL2 is reduced at least 20-fold. In oneembodiment the content of PLBL2 is reduced at least 40-fold. In oneembodiment the content of PLBL2 is reduced at least 50-fold. In oneembodiment the content of PLBL2 is reduced at least 90-fold. In oneembodiment the content of PLBL2 is reduced at least 100-fold. In somecases the level of reduction is even higher. In some embodiments thecontent of PLBL2 is reduced at least 200-fold. In some embodiments thecontent of PLBL2 is reduced at least 250-fold. In some embodiments thecontent of PLBL2 is reduced at least 300-fold. In some embodiments thecontent of PLBL2 is reduced at least 400-fold. In some embodiments thecontent of PLBL2 is reduced at least1000-fold. In one embodiment thecontent of PLBL2 is reduced at least by 50%. In one embodiment thecontent of PLBL2 is reduced at least by 66%. In one embodiment thecontent of PLBL2 is reduced at least by 80%. In one embodiment thecontent of PLBL2 is reduced at least by 90%. In one embodiment thecontent of PLBL2 is reduced at least by 95%. In some embodiments thecontent of PLBL2 is reduced to below 10 ng per mg of antibody. In someembodiments the content of PLBL2 is reduced to below 5 ng per mg ofantibody. In some embodiments the content of PLBL2 is reduced to below 2ng per mg of antibody.

In the methods and the uses as reported herein further wash steps can beemployed with medium and/or high conductivity aqueous solutions. In oneembodiment the low conductivity aqueous solution wash step is precededor succeeded by a high conductivity aqueous solution wash step. In oneembodiment the high conductivity aqueous solution has a conductivityvalue of about 20 mS/cm or higher. In one embodiment the highconductivity aqueous solution has a conductivity value of from about 20mS/cm to about 100 mS/cm. In one embodiment an intermediate wash step isperformed with a medium conductivity aqueous solution between the lowconductivity aqueous solution wash step and the high conductivityaqueous solution wash step. In one embodiment the medium conductivityaqueous solution has a conductivity value of from more than 0.5 mS/cm toless than 20 mS/cm.

It has been found that the host cell protein reducing effect can beimproved when the high or medium conductivity aqueous solution furthercomprises an amino acid. In one embodiment the high or mediumconductivity aqueous solution comprises an amino acid. In one embodimentthe high or medium conductivity aqueous solution comprises Histidine orArginine. In one embodiment the high or medium conductivity aqueoussolution comprises Histidine. In one embodiment the high or mediumconductivity aqueous solution comprises Histidine and Tris.

The methods and the uses as reported herein may include one or morefurther chromatography steps. In one embodiment at least one additionalchromatography method/step is performed. In one embodiment an additionalion exchange chromatography method/step is performed. In one embodimentan additional anion exchange chromatography method/step is performed. Inone embodiment an additional anion exchange chromatography method/stepand an additional cation exchange chromatography method/step areperformed.

It has been found that the use of a hydrophobic interactionchromatography step may be omitted. In one embodiment the use or themethods is without an hydrophobic interaction chromatographymethod/step.

One aspect as reported herein is the use of a low conductivity aqueoussolution in a wash step of a protein A chromatography for reducing thecontent of PLBL2 or Clusterin wherein the protein A chromatography isused to purify an IgG4 or IgG1 isotype, e.g., a human IgG4 or IgG1,antibody and wherein the low conductivity aqueous solution has aconductivity value of about 0.5 mS/cm or less and a pH of about 7 orhigher.

One aspect is the use of a low conductivity aqueous solution in a washstep of a protein A chromatography for reducing the content of PLBL2 orClusterin wherein the protein A chromatography is used to purify a humanIgG4 or IgG1 isotype antibody and wherein the low conductivity aqueoussolution has a conductivity value of about 0.5 mS/cm or less and a pH ofabout 7 or higher. In some embodiments, the antibody is an IgG4 isotypeantibody, e.g., an antibody against P-selectin, or a bispecific antibodyagainst factor IXa and factor X, or an antibody against IL-13, or anantibody against amyloid beta. In some embodiments, the antibody is aIgG1 isotype antibody, e.g., an antibody against Influenza B, or anantibody against VEGF-A, or an antibody against CD22, or a bispecificantibody against HER3 and EGFR, or an antibody against amyloid beta, oran antibody against Her2, or a bispecific antibody against Ang2 andVEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA)and CD3.

In an aspect, the present disclosure provides a method for producing ahuman IgG4 or IgG1 isotype antibody comprising

-   -   a) cultivating a cell comprising a nucleic acid encoding the        human IgG4 or IgG1 isotype antibody,    -   b) recovering the human IgG4 or IgG1 isotype antibody from the        cell or the cultivation medium,    -   c) contacting the human IgG4 or IgG1 isotype antibody with a        protein A affinity chromatography material,    -   d) washing the protein A affinity chromatography material with a        low conductivity aqueous solution,    -   e) recovering the human IgG4 or IgG1 isotype antibody from        affinity chromatography material        and thereby producing the human IgG4 or IgG1 isotype antibody,        wherein the low conductivity aqueous solution has a conductivity        value of about 0.5 mS/cm or less and a pH of about 7 or higher.

In an aspect, the present disclosure provides a method for producing ahuman IgG4 or IgG1 isotype antibody comprising

-   -   a) cultivating a cell comprising a nucleic acid encoding the        human IgG4 or IgG1 isotype antibody,    -   b) recovering the human IgG4 or IgG1 isotype antibody from the        cell or the cultivation medium,    -   c) contacting the human IgG4 or IgG1 isotype antibody with a        protein A affinity chromatography material,    -   d) washing the protein A affinity chromatography material with a        low conductivity aqueous solution,    -   e) recovering the human IgG4 or IgG1 isotype antibody from        affinity chromatography material        and thereby producing the human IgG4 or IgG1 isotype antibody,        wherein the low conductivity aqueous solution has a conductivity        value of about 0.5 mS/cm or less and a pH of about 7 or higher,        and wherein the human IgG4 isotype antibody is an antibody        against P-selectin, or a bispecific antibody against factor IXa        and factor X, or an antibody against IL-13, or an antibody        against amyloid beta and wherein the human IgG1 isotype antibody        is an antibody against Influenza B, or an antibody against        VEGF-A, or an antibody against CD22, or a bispecific antibody        against HER3 and EGFR, or an antibody against amyloid beta, or        an antibody against Her2, or a bispecific antibody against Ang2        and VEGF-A, or a bispecific antibody against carcinoembryonic        antigen (CEA) and CD3.

In an aspect, the present disclosure provides a method for purifying ahuman IgG4 or IgG1 isotype antibody from a sample comprising the stepsof

-   -   a) providing a sample comprising a human IgG4 or IgG1 isotype        antibody,    -   b) purifying the human IgG4 or IgG1 isotype antibody with a        protein A affinity chromatography method/step, comprising        washing the protein A affinity chromatography material with low        conductivity aqueous solution,        wherein the low conductivity aqueous solution has a conductivity        value of about 0.5 mS/cm or less and a pH of about 7 or higher.

In an aspect, the present disclosure provides a method for purifying ahuman IgG4 or IgG1 isotype antibody from a sample comprising the stepsof

-   -   a) providing a sample comprising a human IgG4 or IgG1 isotype        antibody,    -   b) purifying the human IgG4 or IgG1 isotype antibody with a        protein A affinity chromatography method/step, comprising        washing the protein A affinity chromatography material with low        conductivity aqueous solution,        wherein the low conductivity aqueous solution has a conductivity        value of about 0.5 mS/cm or less and a pH of about 7 or higher,        and wherein the human IgG4 isotype antibody is an antibody        against P-selectin, or an antibody against factor IXa and factor        X, or an antibody against IL-13, or an antibody against amyloid        beta and wherein the human IgG1 isotype antibody is an antibody        against Influenza B, or an antibody against VEGF-A, or an        antibody against CD22, or a antibody against HER3 and EGFR, or        an antibody against amyloid beta, or an antibody against Her2,        or a bispecific antibody against Ang2 and VEGF-A, or a        bispecific antibody against carcinoembryonic antigen (CEA) and        CD3.

In an aspect, the present disclosure provides a method for producing ahuman IgG4 isotype antibody comprising

-   -   a) cultivating a cell comprising a nucleic acid encoding the        human IgG4 isotype antibody,    -   b) recovering the human IgG4 isotype antibody from the cell or        the cultivation medium,    -   c) contacting the human IgG4 isotype antibody with a protein A        affinity chromatography material,    -   d) washing the protein A affinity chromatography material with a        low conductivity aqueous solution,    -   e) recovering the human IgG4 isotype antibody from affinity        chromatography material        and thereby producing the human IgG4 isotype antibody,        wherein the low conductivity aqueous solution has a conductivity        value of about 0.5 mS/cm or less and a pH of about 7 or higher,        and wherein the human IgG4 isotype antibody is antibody against        factor IXa and factor X.

In an aspect, the present disclosure provides a method for purifying ahuman IgG4 isotype antibody from a sample comprising the steps of

-   -   a) providing a sample comprising a human IgG4 isotype antibody,    -   b) purifying the human IgG4 isotype antibody with a protein A        affinity chromatography method/step, comprising washing the        protein A affinity chromatography material with low conductivity        aqueous solution,        wherein the low conductivity aqueous solution has a conductivity        value of about 0.5 mS/cm or less and a pH of about 7 or higher,        and wherein the human IgG4 isotype antibody is an antibody        against factor IXa and factor X.

The terms “anti-P-selectin antibody” and “an antibody that binds toP-selectin” or “antibody against P-selectin”refer to an antibody that iscapable of binding P-selectin with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingP-selectin. In one embodiment, the extent of binding of ananti-P-selectin antibody to an unrelated, non-P-selectin protein is lessthan about 10% of the binding of the antibody to P-selectin as measured,e.g., by ELISA or surface plasmon resonance. In certain embodiments, ananti-P-selectin antibody binds to an epitope of P-selectin that isconserved among P-selectin from different species. The above also holdsfor the terms “antibody against factor IXa and factor X” or “antibodyagainst IL-13” or “antibody against amyloid beta” or the like.

In some embodiments, the antibody against P-selectin is inclacumab (IgG4isotype) as described in WO 2005/100402 or SEQ ID NO: 07 to 12. In someembodiments, the antibody is a bispecific antibody against factor IXaand factor X, e.g., anti-FIXa/X antibody (IgG4 isotype) as described inWO 2012/067176. In some embodiments, the antibody is an antibody againstHer2, e.g., trastuzumab (IgG1 isotype) as described in WO 1992/022653.In some embodiments, the antibody is a bispecific antibody againstangiopoietin 2 (Ang2) and vascular endothelial growth factor A (VEGF-A),e.g., vanucizumab (IgG1 isotype) as described in WO 2011/117329 or SEQID NO: 01 to 04. In some embodiments, the antibody is an antibodyagainst amyloid beta, e.g., gantenerumab (IgG1 isotype) as described inWO 2003/070760 or SEQ ID NO: 05 to 06, or crenezumab (IgG4 isotype). Insome embodiments, the antibody is an antibody against CD22, an antibodyagainst IL13 (e.g., lebrikizumab), a bispecific antibody against Her3and EGFR (e.g., duligotuzumab), an antibody against VEGF-A (e.g.,bevacizumab), and an antibody against Influenza B. The terms VEGF orVEGF-A can be used interchangeably herein.

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen in an in-vitroassay, preferably in a surface plasmon resonance assay (SPR, BIAcore,GE-Healthcare Uppsala, Sweden). The affinity of the binding is definedby the terms ka (rate constant for the association of the antibody fromthe antibody/antigen complex), k_(d) (dissociation constant), and K_(D)(k_(d)/k_(a)). Binding or specifically binding means a binding affinity(K_(D)) of 10⁻⁷ mol/L or less.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments. A Fab fragment is an antibody fragment obtained by a papaindigestion of a (full length/complete) antibody.

Bispecific antibodies” are antibodies which have two differentantigen-binding specificities. The term “bispecific” antibody as usedherein denotes an antibody that has at least two binding sites each ofwhich bind to different epitopes.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “human IgG isotype antibody” denotes an antibody that comprisesa constant region that is derived from a human wild-type IgG isotype,i.e. for example it may comprise a constant region derived from a humanIgG isotype with a mutation, e.g. an P329G mutation (numbering accordingto Kabat).

The term “human IgG4 isotype antibody” denotes an antibody thatcomprises a constant region that is derived from a human wild-type IgG4isotype, i.e. for example it may comprise a constant region derived froma human IgG4 isotype with a mutation, e.g. an an P329G mutation and/orS228P, L235E mutation (numbering according to Kabat).

The term “Fc-region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc-regions andvariant Fc-regions. In one embodiment, a human IgG heavy chain Fc-regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) or the C-terminalglycyl-lysine dipeptide (Gly446Lys447) of the Fc-region may or may notbe present. Unless otherwise specified herein, numbering of amino acidresidues in the Fc-region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat, E. A.et al., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991), NIHPublication 91-3242.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.The term “cell” includes cells which are used for the expression ofnucleic acids. In one embodiment the host cell is a CHO cell (e.g. CHOK1, CHO DG44), or a BHK cell, or a NS0 cell, or a SP2/0 cell, or a HEK293 cell, or a HEK 293 EBNA cell, or a PER.C6® cell, or a COS cells. Inanother embodiment the cell is a CHO cell, or a BHK cell, or a PER.C6®cell. As used herein, the expression “cell” includes the subject celland its progeny.

The term “washing” denotes the applying of a solution to an affinitychromatography material in order to remove non specifically boundpolypeptides and non-polypeptide compounds from the chromatographymaterial, especially to remove host cell protein and host cell DNA. Theterm “washing” does not encompass the elution of bound material from anaffinity chromatography material.

Different methods are well established and widespread used for proteinrecovery and purification, such as affinity chromatography withmicrobial proteins (e.g. protein A or protein G affinity chromatography)affinity chromatographie with a recombinant protein as ligand (e.g.single chain Fv as ligand, e.g. Kappa select), ion exchangechromatography (e.g. cation exchange (carboxymethyl resins), anionexchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis). These methods can be combined independently indifferent embodiments as reported herein.

The term “protein A” denotes a protein A polypeptide either obtainedfrom a natural source or produced synthetically.

The term “protein A chromatography material” denotes an inert solidphase to which a protein A is covalently linked.

In one embodiment the protein A chromatography material is selected fromMabSelectSure, ProSep vA, Mab Capture A, ProSep Ultra Plus, Mab Select,Mab Select Xtra, Poros A, or ProSep A.

The term “high conductivity aquaeous solution” denotes an aquaeoussolution with a high conductivity value. The conductivity value may beabout 20 mS/cm or higher.

The term “medium conductivity aquaeous solution” denotes an aquaeoussolution with a medium conductivity value. The conductivity value may bemore than 0.5 mS/cm to less than 20 mS/cm.

The term “low conductivity aquaeous solution” denotes an aquaeoussolution with a low conductivity value. The conductivity value may beabout 0.5 mS/cm or less. The conductivity value may be about 1.2 mS/cmor less, if the pH is about 8.5 or higher. The conductivity values canbe determined by standard methods known to the person skilled in theart.

The following examples and sequences are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

Specific Embodiments of the Invention

-   1. Use of a low conductivity aqueous solution in a wash step of an    affinity chromatography for reducing the content of a host cell    protein.-   2. Use according to embodiment 1, wherein the affinity    chromatography is used to purify a human IgG isotype antibody.-   3. Use according to embodiment 2, wherein the affinity    chromatography is used to purify a human IgG4 isotype antibody or a    human IgG1 isotype antibody.-   4. Use according to embodiment 3, wherein the affinity    chromatography is used to purify a human IgG4 isotype antibody or a    human IgG1 isotype antibody without a glycosylated glycosylation    site in its Fab fragment/with exactly one glycosylation site (at    position Asn 297 numbering according to Kabat).-   5. Use according to embodiment 4, wherein the low conductivity    aqueous solution has a conductivity value of about 0.5 mS/cm or    less.-   6. Use according to embodiment 5, wherein the low conductivity    aqueous solution has a conductivity value of from about 0.03 μS/cm    to about 0.5 mS/cm.-   7. Use according to embodiment 5, wherein the low conductivity    aqueous solution has a conductivity value of from about 0.05 μS/cm    to about 0.35 mS/cm.-   8. Use according to any of embodiments 5 to 7, wherein the low    conductivity aqueous solution is not deionized water.-   9. Use according to any of the previous embodiments, wherein the    affinity chromatography is a protein A affinity chromatography or a    Protein G affinity chromatography or a single chain Fv ligand    (KappaSelect) affinity chromatography.-   10. Use according to embodiment 9, wherein the affinity    chromatography is a protein A affinity chromatography.-   11. Use according to embodiment 10, wherein the protein A affinity    chromatography is selected from the group comprising MabSelectSure    affinity chromatography, ProSep vA affinity chromatography, Poros    Mab Capture A affinity chromatography, ProSep Ultra Plus affinity    chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl    AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF).-   12. Use according to any one of the previous embodiments, wherein    said host cell protein is a Chinese hamster ovary (CHO) host cell    protein.-   13. Use according to embodiment 12, wherein the host cell protein is    a phospholipase.-   14. Use according to embodiment 13, wherein the host cell protein is    a phospholipase A, phospholipase B, phospholipase C or phospholipase    D.

15. Use according to embodiments 12, 13 or 14, wherein the host cellprotein is phospholipase B-like 2 (PLBL2)

-   16. Use according to embodiment 12, wherein the host cell protein is    phospholipase B-like 2 (PLBL2) or Clusterin.-   17. Use according to any of the preceding embodiments, wherein the    low conductivity aqueous solution contains    tris(hydroxymethyl)aminomethane (Tris).-   18. Use according to embodiment 17, wherein the low conductivity    aqueous solution comprises about 0.1 mM to about 10 mM Tris.-   19. Use according to embodiment 18, wherein the low conductivity    aqueous solution comprises about 0.1 mM to about 8 mM Tris.-   20. Use according to embodiment 19, wherein the low conductivity    aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.-   21. Use according to embodiment 20, wherein the low conductivity    aqueous solution comprises about 2 mM Tris.-   22. Use according to any one of embodiments 17 to 21, wherein the    low conductivity aqueous solution contains potassium phosphate.-   23. Use according to embodiment 22, wherein the low conductivity    aqueous solution comprises about 0.05 mM to about 5 mM potassium    phosphate.-   24. Use according to embodiment 23, wherein the low conductivity    aqueous solution comprises about 0.05 mM to about 2 mM potassium    phosphate.-   25. Use according to embodiment 24, wherein the low conductivity    aqueous solution comprises about 0.5 mM potassium phosphate.-   26. Use according to any of the preceeding embodiments, wherein the    low conductivity aqueous solution has a pH of about 7 or higher.-   27. Use according to embodiment 26, wherein the low conductivity    aqueous solution has a pH of about 7.5 or higher.-   28. Use according to embodiment 27, wherein the low conductivity    aqueous solution has a pH of from about 7 to about 9.5.-   29. Use according to embodiment 28, wherein the low conductivity    aqueous solution has a pH of from about 7.5 to about 8.5.-   30. Use according to embodiment 29, wherein the low conductivity    aqueous solution has a pH of about 8.-   31. Use according to any one of the preceeding embodiments, wherein    the low conductivity aqueous solution wash step is preceded or    succeeded by a high conductivity aqueous solution wash step.-   32. Use according to embodiment 31, wherein the low conductivity    aqueous solution wash step is preceded by a high conductivity    aqueous solution wash step.-   33. Use according to embodiment 31, wherein the high conductivity    aqueous solution has a conductivity value of about 20 mS/cm or    higher.-   34. Use according to embodiment 33, wherein the high conductivity    aqueous solution has a conductivity value of from about 20 mS/cm to    about 100 mS/cm.-   35. Use according to embodiment 31, wherein an intermediate wash    step is performed with a medium conductivity aqueous solution    between the low conductivity aqueous solution wash step and the high    conductivity aqueous solution wash step.-   36. Use according to embodiment 35, wherein the medium conductivity    aqueous solution has a conductivity value of from more than 0.5    mS/cm to less than 20 mS/cm.-   37. Use according to any one of embodiments 33 to 36, wherein the    high or medium conductivity aqueous solution comprises an amino    acid.-   38. Use according to embodiment 37, wherein the high or medium    conductivity aqueous solution comprises Histidine.-   39. Use according to embodiment 37, wherein the high or medium    conductivity aqueous solution comprises Histidine and Tris.-   40. Use according to any of the preceeding embodiments, wherein at    least one additional chromatography method/step is performed.-   41. Use according to embodiment 40, wherein an additional ion    exchange chromatography method/step is performed.-   42. Use according to embodiment 41, wherein an additional anion    exchange chromatography method/step is performed.-   43. Use according to embodiment 40, wherein an additional cation    exchange chromatography method/step is performed.-   44. Use according to embodiment 40, wherein an additional anion    exchange chromatography method/step and an additional cation    exchange chromatography method/step are performed.-   45. Use according to embodiment 40, wherein the use is without a    hydrophobic interaction chromatography method/step.-   46. Use according to any of the preceeding embodiments, wherein the    human IgG4 isotype antibody is an antibody against P-selectin or an    antibody against factor IXa and factor X or an antibody against    IL-13 or an antibody against amyloid beta.-   47. Use according to embodiments 1 to 45, wherein the human IgG1    isotype antibody is an antibody against Influenza B or an antibody    against VEGF-A or an antibody against CD22 or an antibody against    HER3 and EGFR or an antibody against amyloid beta or an antibody    against Her2 or an antibody against Ang2 and VEGF-A or an antibody    against carcinoembryonic antigen (CEA) and CD3.-   48. Method for producing a human IgG isotype antibody comprising    -   a) cultivating a cell comprising a nucleic acid encoding the        human IgG isotype antibody,    -   b) recovering the human IgG isotype antibody from the cell or        the cultivation medium,    -   c) contacting the human IgG isotype antibody with an affinity        chromatography material,    -   d) washing the affinity chromatography material with a low        conductivity aqueous solution    -   e) recovering the human IgG isotype antibody from affinity        chromatography material        and thereby producing the human IgG isotype antibody.-   49. Method for producing a human IgG4 isotype antibody comprising    -   a) cultivating a cell comprising a nucleic acid encoding a human        IgG4 isotype antibody,    -   b) recovering the human IgG4 isotype antibody from the cell or        the cultivation medium,    -   c) contacting the human IgG4 isotype antibody with an affinity        chromatography material,    -   d) washing the affinity chromatography material with a low        conductivity aqueous solution    -   e) recovering the human IgG4 isotype antibody from the affinity        chromatography material        and thereby producing the human IgG4 isotype antibody.-   50. Method according to embodiment 48, wherein the human IgG isotype    antibody is a human IgG4 isotype antibody or a human IgG1 isotype    antibody.-   51. Method according to embodiment 48, wherein the human IgG isotype    antibody is a human IgG4 isotype antibody or a human IgG1 isotype    antibody without a glycosylated glycosylation site in its Fab    fragment/with exactly one glycosylation site (at position Asn 297    numbering according to Kabat).-   52. Method according to embodiment 48, wherein the low conductivity    aqueous solution has a conductivity value of about 0.5 mS/cm or    less.-   53. Method according to embodiment 52, wherein the low conductivity    aqueous solution has a conductivity value of from about 0.03 μS/cm    to about 0.5 mS/cm.-   54. Method according to embodiment 53, wherein the low conductivity    aqueous solution has a conductivity value of from about 0.05 μS/cm    to about 0.35 mS/cm.-   55. Method according to embodiment 54, wherein the low conductivity    aqueous solution is not deionized water.-   56. Method according to any one of embodiments 48 to 55, wherein the    affinity chromatography is a protein A affinity chromatography or a    Protein G affinity chromatography or a single chain Fv ligand    (KappaSelect) affinity chromatography.-   57. Method according to embodiment 56, wherein the affinity    chromatography is a protein A affinity chromatography.-   58. Method according to embodiment 56, wherein the protein A    affinity chromatography is selected from the group comprising    MabSelectSure affinity chromatography, ProSep vA affinity    chromatography, Poros Mab Capture A affinity chromatography, ProSep    Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect,    Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A    HC-650HF).-   59. Method according to embodiment 48 or 49, wherein the content of    a host cell protein is reduced.-   60. Method according to any one of embodiments 59, wherein said host    cell protein is a Chinese hamster ovary (CHO) host cell protein.-   61. Method according to embodiment 60, wherein the host cell protein    is a phospholipase.-   62. Method according to embodiment 61, wherein the host cell protein    is a phospholipase A, phospholipase B, phospholipase C or    phospholipase D.-   63. Method according to any one of embodiments 62, wherein the host    cell protein is phospholipase B-like 2 (PLBL2).-   64. Method according to embodiment 60, wherein the host cell protein    is phospholipase B-like 2 (PLBL2) or Clusterin.-   65. Method according to any of embodiments 48 to 64, wherein the low    conductivity aqueous solution contains    tris(hydroxymethyl)aminomethane (Tris).-   66. Method according to embodiment 65, wherein the low conductivity    aqueous solution comprises about 0.1 mM to about 10 mM Tris.-   67. Method according to embodiment 66, wherein the low conductivity    aqueous solution comprises about 0.1 mM to about 8 mM Tris.-   68. Method according to embodiment 67, wherein the low conductivity    aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.-   69. Method according to embodiment 68, wherein the low conductivity    aqueous solution comprises about 2 mM Tris.-   70. Method according to embodiment 65 to 69, wherein the low    conductivity aqueous solution contains potassium phosphate.-   71. Method according to embodiment 70, wherein the low conductivity    aqueous solution comprises about 0.2 mM to about 5 mM potassium    phosphate.-   72. Method according to embodiment 71, wherein the low conductivity    aqueous solution comprises about 0.05 mM to about 2 mM potassium    phosphate.-   73. Method according to embodiment 72, wherein the low conductivity    aqueous solution comprises about 0.5 mM potassium phosphate.-   74. Method according to any of embodiments 48 to 73, wherein the low    conductivity aqueous solution has a pH of about 7 or higher.-   75. Method according to embodiment 74, wherein the low conductivity    aqueous solution has a pH of about 7.5 or higher.-   76. Method according to embodiment 75, wherein the low conductivity    aqueous solution has a pH of from about 7 to about 9.5.-   77. Method according to embodiment 76, wherein the low conductivity    aqueous solution has a pH of from about 7.5 to about 8.5.-   78. Method according to embodiment 77, wherein the low conductivity    aqueous solution has a pH of about 8.-   79. Method according to embodiment 48 or 49, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution before or after washing    the affinity chromatography material with low conductivity aqueous    solution.-   80. Method according to embodiment 79, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution before washing the    affinity chromatography material with low conductivity aqueous    solution.-   81. Method according to embodiment 79, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution and/or with a medium    conductivity aqueous solution before or after washing the affinity    chromatography material with low conductivity aqueous solution.-   82. Method according to embodiment 79, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution and/or with a medium    conductivity aqueous solution before washing the affinity    chromatography material with low conductivity aqueous solution.-   83. Method according to any one of embodiments 79 to 82, wherein the    high conductivity aqueous solution has a conductivity value of about    20 mS/cm or higher.-   84. Method according to embodiment 83, wherein the high conductivity    aqueous solution has a conductivity value of from about 20 mS/cm to    about 100 mS/cm.-   85. Method according to any one of embodiments 81 to 82, wherein the    medium conductivity aqueous solution has a conductivity value of    from more than 0.5 mS/cm to less than 20 mS/cm.-   86. Method according to any one of embodiments 79 to 85, wherein the    high or medium conductivity aqueous solution comprises an amino    acid.-   87. Method according to embodiment 86, wherein the high or medium    conductivity aqueous solution comprises Histidine.-   88. Method according to embodiment 86 or 87, wherein the high or    medium conductivity aqueous solution comprises Histidine and Tris.-   89. Method according to embodiment 48 or 49, wherein at least one    additional chromatography method/step is performed after step e).-   90. Method according to embodiment 89, wherein an additional ion    exchange chromatography method/step is performed after step e).-   91. Method according to embodiment 90, wherein an additional anion    exchange chromatography method/step is performed after step e).-   92. Method according to embodiment 90, wherein an additional cation    exchange chromatography method/step is performed after step e).-   93. Method according to embodiment 90, wherein an additional anion    exchange chromatography method/step and an additional cation    exchange chromatography method/step are performed after step e).-   94. Method according to embodiment 48 or 49, wherein the method is    without an hydrophobic interaction chromatography method/step.-   95. Method according to any of embodiments 49 to 94, wherein the    human IgG4 isotype antibody is an antibody against P-selectin or an    antibody against factor IXa and factor X or an antibody against    IL-13 or an antibody against amyloid beta.-   96. Method according to any one of embodiments 48 or 50 to 94,    wherein the human IgG1 isotype antibody is an antibody against    Influenza B or an antibody against VEGF-A or an antibody against    CD22 or a bispecific antibody against HER3 and EGFR or an antibody    against amyloid beta or an antibody against Her2 or a bispecific    antibody against Ang2 and VEGF-A or an antibody against    carcinoembryonic antigen (CEA) and CD3.-   97. Method for purifying a human IgG isotype antibody from a sample    comprising the steps of    -   a) providing a sample comprising a human IgG isotype antibody,    -   b) purifying the human IgG isotype antibody with a affinity        chromatography method/step, comprising washing the affinity        chromatography material with low conductivity aqueous solution.-   98. Method according to embodiment 97, wherein the human IgG isotype    antibody is a human IgG4 isotype antibody or a human IgG1 isotype    antibody.-   99. Method according to embodiment 98, wherein the human IgG isotype    antibody is a human IgG4 isotype antibody or a human IgG1 isotype    antibody without a glycosylated glycosylation site in its Fab    fragment/with exactly one glycosylation site (at position Asn 297    numbering according to Kabat).-   100. Method according to any one of embodiments 97 to 99, wherein    the low conductivity aqueous solution has a conductivity value of    about 0.5 mS/cm or less.-   101. Method according to embodiment 100, wherein the low    conductivity aqueous solution has a conductivity value of from about    0.03 μS/cm to about 0.5 mS/cm.-   102. Method according to embodiment 101, wherein the low    conductivity aqueous solution has a conductivity value of from about    0.05 μS/cm to about 0.35 mS/cm.-   103. Method according to embodiment 102, wherein the low    conductivity aqueous solution is not deionized water.-   104. Method according to any one of embodiments 97 to 104, wherein    the affinity chromatography is a protein A affinity chromatography    or a Protein G affinity chromatography or a single chain Fv ligand    (KappaSelect) affinity chromatography.-   105. Method according to embodiment 104, wherein the affinity    chromatography is a protein A affinity chromatography.-   106. Method according to embodiment 105, wherein the protein A    affinity chromatography is selected from the group comprising    MabSelectSure affinity chromatography, ProSep vA affinity    chromatography, Poros Mab Capture A affinity chromatography, ProSep    Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect,    Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A    HC-650HF).-   107. Method according to any one of embodiments 97 to 106, wherein    the content of a host cell protein is reduced.-   108. Method according to embodiment 107, wherein said host cell    protein is a Chinese hamster ovary (CHO) host cell protein.-   109. Method according to embodiment 108, wherein the host cell    protein is a phospholipase.-   110. Method according to embodiment 109, wherein the host cell    protein is a phospholipase A, phospholipase B, phospholipase C or    phospholipase D.-   111. Method according to any one of embodiments 110, wherein the    host cell protein is phospholipase B-like 2 (PLBL2).-   112. Method according to embodiment 107, wherein the host cell    protein is phospholipase B-like 2 (PLBL2) or Clusterin.-   113. Method according to any one of embodiments 97 to 112, wherein    the low conductivity aqueous solution contains    tris(hydroxymethyl)aminomethane (Tris).-   114. Method according to embodiment 113, wherein the low    conductivity aqueous solution comprises about 0.1 mM to about 10 mM    Tris.-   115. Method according to embodiment 114, wherein the low    conductivity aqueous solution comprises about 0.1 mM to about 8 mM    Tris.-   116. Method according to embodiment 115, wherein the low    conductivity aqueous solution comprises about 0.5 mM to about 6.5 mM    Tris.-   117. Method according to embodiment 116, wherein the low    conductivity aqueous solution comprises about 2 mM Tris.-   118. Method according to any one of embodiments 113 to 117, wherein    the low conductivity aqueous solution contains potassium phosphate.-   119. Method according to embodiment 118, wherein the low    conductivity aqueous solution comprises about 0.2 mM to about 5 mM    potassium phosphate.-   120. Method according to embodiment 119, wherein the low    conductivity aqueous solution comprises about 0.2 mM to about 2 mM    potassium phosphate.-   121. Method according to embodiment 120, wherein the low    conductivity aqueous solution comprises about 0.5 mM potassium    phosphate.-   122. Method according to any one of embodiments 97 to 121, wherein    the low conductivity aqueous solution has a pH of about 7 or higher.-   123. Method according to embodiment 122, wherein the low    conductivity aqueous solution has a pH of about 7.5 or higher.-   124. Method according to embodiment 123, wherein the low    conductivity aqueous solution has a pH of from about 7 to about 9.5.-   125. Method according to embodiment 124, wherein the low    conductivity aqueous solution has a pH of from about 7.5 to about    8.5.-   126. Method according to embodiment 125, wherein the low    conductivity aqueous solution has a pH of about 8.-   127. Method according to any one of embodiments 97 to 126, wherein    the method additionally comprises washing the affinity    chromatography material with a high conductivity aqueous solution    before or after washing the affinity chromatography material with    low conductivity aqueous solution.-   128. Method according to embodiment 127, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution before washing the    affinity chromatography material with low conductivity aqueous    solution.-   129. Method according to embodiment 127, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution and/or with a medium    conductivity aqueous solution before or after washing the affinity    chromatography material with low conductivity aqueous solution.-   130. Method according to embodiment 127, wherein the method    additionally comprises washing the affinity chromatography material    with a high conductivity aqueous solution and/or with a medium    conductivity aqueous solution before washing the affinity    chromatography material with low conductivity aqueous solution.-   131. Method according to any one of embodiments 127 to 130, wherein    the high conductivity aqueous solution has a conductivity value of    about 20 mS/cm or higher.-   132. Method according to embodiment 131, wherein the high    conductivity aqueous solution has a conductivity value of from about    20 mS/cm to about 100 mS/cm.-   133. Method according to embodiment 129 or 130, wherein the medium    conductivity aqueous solution has a conductivity value of from more    than 0.5 mS/cm to less than 20 mS/cm.-   134. Method according to any one of embodiments 127 to 133, wherein    the high or medium conductivity aqueous solution comprises an amino    acid.-   135. Method according to embodiment 134, wherein the high or medium    conductivity aqueous solution comprises Histidine.-   136. Method according to embodiment 134, wherein the high or medium    conductivity aqueous solution comprises Histidine and Tris.-   137. Method according to embodiment 97 or 98, wherein at least one    additional chromatography method/step is performed after step b).-   138. Method according to embodiment 137, wherein an additional ion    exchange chromatography method/step is performed after step b).-   139. Method according to embodiment 138, wherein an additional anion    exchange chromatography method/step is performed after step b).-   140. Method according to embodiment 138, wherein an additional    cation exchange chromatography method/step is performed after step    b).-   141. Method according to embodiment 138, wherein an additional anion    exchange chromatography method/step and an additional cation    exchange chromatography method/step are performed after step b).-   142. Method according to any one of embodiments 97 to 141, wherein    the method is without an hydrophobic interaction chromatography    method/step.-   143. Method according to embodiment 97 or 98, wherein the human IgG4    isotype antibody is an antibody against P-selectin or an antibody    against factor IXa and factor X or an antibody against IL-13 or an    antibody against amyloid beta.-   144. Method according to embodiment 97 or 98, wherein the human IgG1    isotype antibody is an antibody against Influenza B or an antibody    against VEGF-A or an antibody against CD22 or an (bispecific)    antibody against HER3 and EGFR or an antibody against amyloid beta    or an antibody against Her2 or a bispecific antibody against Ang2    and VEGF-A or a bispecific antibody against carcinoembryonic antigen    (CEA) and CD3.-   145. Use of a low conductivity aqueous solution in a wash step of a    protein A chromatography for reducing the content of a host cell    protein wherein the protein A chromatography is used to purify a    human IgG4 or IgG1 isotype antibody, wherein the low conductivity    aqueous solution has a conductivity value of about 0.5 mS/cm or    less.-   146. Method for producing a human IgG4 or IgG1 isotype antibody    comprising the steps of    -   a) cultivating a cell comprising a nucleic acid encoding a human        IgG4 or IgG1 isotype antibody,    -   b) recovering the human IgG4 or IgG1 isotype antibody from the        cell or the cultivation medium,    -   c) contacting the human IgG4 or IgG1 isotype antibody with a        protein A chromatography material,    -   d) washing the protein A chromatography material with a low        conductivity aqueous solution, wherein the low conductivity        aqueous solution has a conductivity value of about 0.5 mS/cm or        less,    -   e) recovering the human IgG4 or IgG1 isotype antibody from the        protein A chromatography material        and thereby producing the human IgG4 or IgG1 isotype antibody.-   147. Method for purifying a human IgG4 or IgG1 isotype antibody from    a sample comprising the steps of    -   a) providing a sample comprising a human IgG4 or IgG1 isotype        antibody,    -   b) purifying the human IgG4 or IgG1 isotype antibody with a        protein A chromatography method/step, comprising washing the        protein A chromatography material with a low conductivity        aqueous solution, wherein the low conductivity aqueous solution        has a conductivity value of about 0.5 mS/cm or less.-   148. Use according to any of embodiments 5 to 7, wherein the low    conductivity aqueous solution is deionized water.-   149. Method according to embodiment 54, wherein the low conductivity    aqueous solution is deionized water.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 01 variable heavy chain domain VH of <VEGF>SEQ ID NO: 02 variable light chain domain VL of <VEGF>SEQ ID NO: 03 variable heavy chain domain VH of <ANG-2>SEQ ID NO: 04 variable light chain domain VL of <ANG-2>SEQ ID NO: 05 variable heavy chain domain VH of anti-amyloid betaantibody (IgG1 isotype)SEQ ID NO: 06 variable light chain domain VL of anti-amyloid betaantibody (IgG1 isotype)SEQ ID NO: 07 variable heavy chain domain VH1 of anti-P-selectinantibodySEQ ID NO: 08 variable heavy chain domain VH2 of anti-P-selectinantibodySEQ ID NO: 09 variable heavy chain domain VH3 of anti-P-selectinantibodySEQ ID NO: 10 variable light chain domain VL1 of anti-P-selectinantibodySEQ ID NO: 11 variable light chain domain VL2 of anti-P-selectinantibodySEQ ID NO: 12 variable light chain domain VL3 of anti-P-selectinantibody

EXAMPLE 1 Material and Methods Antibodies

The current invention is exemplified using a number of exemplaryantibodies, including: an antibody against P-selectin (anti-P-selectinantibody; inclacumab; IgG4 isotype) as described in WO 2005/100402 orSEQ ID NO: 07 to SEQ ID NO: 12; a bispecific antibody against factor IXaand factor X (anti-FIXa/X antibody; IgG4 isotype) as described in WO2012/067176; with an antibody against Her2; a bispecific antibodyagainst Ang2 and VEGF-A (anti-Ang2/VEGF-A antibody; vanucizumab; IgG1isotype) as described in WO 2011/117329 or SEQ ID NO: 01 to SEQ ID NO:04; an antibody against amyloid beta (anti-amyloid beta antibody;gantenerumab; IgG1 isotype) as described in WO 2003/070760 or SEQ ID NO:05 to SEQ ID NO: 06. Also included herein are a number of IgG1antibodies and IgG4 antibodies, as described in the examples below.

Detection Methods for Overall Host Cell Protein (HCP), PhospholipaseB-like 2 Protein (PLBL2) and Clusterin

-   a) CHO HCP Assay

The residual CHO HCP content in process samples is determined by anelectrochemiluminescence immunoassay (ECLIA) on cobas e 411 immunoassayanalyzer (Roche Diagnostics).

The assay is based on a sandwich principle using polyclonal anti-CHO HCPantibody from sheep.

First incubation: Chinese hamster ovary host cell protein (CHO HCP) from15 μL sample (neat and/or diluted) and a biotin conjugated polyclonalCHO HCP specific antibody form a sandwich complex, which becomes boundto streptavidin-coated microparticles via interaction of biotin withstreptavidin.

Second incubation: After addition of polyclonal CHO HCP-specificantibody labeled with ruthenium complex(Tris(2,2′-bipyridyl)ruthenium(II)-complex) a ternary sandwich complexis formed on the microparticles.

The reaction mixture is aspirated into the measuring cell where themicroparticles are magnetically captured onto the surface of theelectrode. Unbound substances are then removed in a washing step.Application of a voltage to the electrode then induces chemiluminescentemission which is measured by a photomultiplier.

The concentration of CHO HCP in the test sample is finally calculatedfrom a CHO

HCP standard curve of known concentration.

-   b) CHO PLBL2 Assay

The residual Chinese hamster ovary (CHO) Phospholipase B-like 2 protein(PLBL2) content in process samples is determined by anelectrochemiluminescence immunoassay (ECLIA) on cobas e 411 immunoassayanalyzer (Roche Diagnostics).

The assay is based on a sandwich principle using monoclonal anti-CHOPLBL2 antibody from mouse.

In a first incubation step, CHO PLBL2 from 30 μL sample (neat and/ordiluted), biotin labeled monoclonal CHO PLBL2-specific antibody, and amonoclonal CHO PLBL2-specific antibody labeled with a ruthenium complex(Tris(2,2′-bipyridyl)ruthenium(II)-complex) form a sandwich complex.

In a second step after addition of streptavidin-coated microparticles,the ternary complex becomes bound to the solid phase via interaction ofbiotin and streptavidin.

The reaction mixture is aspirated into the measuring cell where themicroparticles are magnetically captured onto the surface of theelectrode. Unbound substances are then removed in a washing step.Application of a voltage to the electrode then induceschemiluminescence, which is measured by a photomultiplier.

The concentration of CHO PLBL2 in the test sample is finally calculatedfrom a CHO PLBL2 standard curve of known concentration.

-   c) Clusterin Assay

The residual Clusterin content in process samples is determined by acommercial assay from Merck Millipore (GyroMark HT Kit GYRCLU-37K) whichwas used according to the manufacturer's instructions.

In brief, this assay is a Sandwich ELISA based, sequentially, on:

-   1) binding of the rat Clusterin biotinylated capture antibody to the    streptavidin coated affinity columns of the Bioaffy 1000 nL CD,-   2) capture of rat Clusterin molecules from samples to the anti    Clusterin antibody,-   3) binding of a second dye-labeled anti Clusterin detection antibody    to the captured molecules,-   4) quantification of the rat Clusterin using the Gyrolab Evaluator.

EXAMPLE 2 Purification of an Anti-P-Selectin Antibody (IgG4 Isotype) ina Protein A Chromatography Antibody: Anti-P-Selectin GeneralChromatography Conditions

Column resin: Protein A material “Mab Select SuRe” (GE-Healthcare) Ø1cm, Height: 20.1 cm, CV: 15.79 ml

Equipment: Äkta Avant 150

Flow rate: 300 cm/h during all steps

A solution containing an anti-P-Selectin antibody, was applied to aProtein A affinity column after equilibration (step 1) of the column.Initial load of PLBL2 determined in solution containing ananti-P-Selectin antibody: 335 ng PLBL2/mg of antibody. Initial load ofClusterin determined in solution containing an anti-P-Selectin antibody:2874.8 ng Clusterin/mg of antibody. Initial load of CHOP determined insolution containing an anti-P-Selectin antibody: 100971 ng CHOP/mg ofantibody.

The chromatographic steps were performed according to the followinggeneral scheme:

Step 1: Equilibration:

Step 2: Load of antibody containing solution

Step 3: Wash I Step 4: Wash II Step 5: Wash III

Step 6: Wash IV (additional wash)

Step 7: Elution

After Elution from Protein A affinity column the protein was determinedby size exclusion chromatography (SEC) and spectrophotometrically (OD)Analytics.

SEC:

-   -   Resin: TSK 3000 (Tosoh)    -   Column: 300×7.8 mm    -   Flow rate: 0.5 ml/min    -   Buffer: 200 mM potassium phosphate containing    -    250 mM potassium chloride, adjusted to pH 7.0    -   Wavelength: 280 nm

OD:

-   -   Specific coefficient: 1.54    -   Wavelength: 280 nm minus 320 nm

Specific buffer conditions for Protein A chromatography (anti-P-Selectinantibody)

a) Control (wash with equilibration buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0b) low conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0c) low conductivity wash (with potassium phosphate (KP) only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0

Step 6: Wash IV: 0.5 mM potassium phosphate, pH 8.0Step 7: Elution: 50 mM acetic acid, pH 4.0d) high conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0e) low conductivity wash (with Tris buffer only; pH 6.0)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step6: Wash IV: 2 mM Tris, pH 6.0

Step 7: Elution: 50 mM acetic acid, pH 4.0f) high conductivity wash (with Histidine (His)/Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0 Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0g) low conductivity Tris +high conductivity Histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0 Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0h) low conductivity potassium phosphate (KP)+high conductivity Histidine(His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0

Step 6: Wash IV: 0.5 mM potassium phosphate, pH 8.0Step 7: Elution: 50 mM acetic acid, pH 4.0i) low conductivity Tris+high conductivity Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0j) low conductivity Tris; pH 6.0+high conductivity Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:2 mM Tris, pH 6.0

Step 7: Elution: 50 mM acetic acid, pH 4.0

Results:

HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 218105.9 27.4 93.44 b 105 0.8 11.2 94.61 c 114 0.7 11.8 86.67 d 48 14.723.2 89.33 e 155 18.7 53.8 107.3 f 106 2.9 21.6 84.9 g 83 0.4 11.8 85 h91 0.4 9.1 80.34 i 90 0.4 15.7 84.92 j 141 1.5 53 106.9

EXAMPLE 3 Purification of an Anti-Amyloid Beta Antibody (IgG1 Isotype)in a Protein A Chromatography

General conditions were according to the conditions described in Example2.

Antibody: anti-amyloid beta.

Initial load of PLBL2 determined in solution containing an anti-amyloidbeta antibody: 2019.7 ng PLBL2/mg of antibody. Initial load of CHOPdetermined in solution containing an anti-amyloid beta antibody: 578908ng CHOP/mg of antibody.

Specific buffer conditions for Protein A chromatography

a) Control (wash with equilibration buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0b) low conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0c) high conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0d) low conductivity Tris+high conductivity Histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0 Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0

HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 682817.3 n.d. 80.3 b 7794 17.8 n.d. 73.1 c 1595 1.7 n.d. 55.6 d 6132 2.3n.d. 67.3

EXAMPLE 4 Purification of an Anti-Her2 Antibody (IgG1 Isotype) in aProtein A Chromatography

General conditions were according to the conditions described in Example2.

Antibody: anti-Her2

Initial load of PLBL2 determined in solution containing an anti-Her2antibody: 1662.5 ng PLBL2/mg of antibody. Initial load of CHOPdetermined in solution containing an anti-Her2 antibody: 727070 ngCHOP/mg of antibody.

Specific buffer conditions for Protein A chromatography

a) Control (wash with equilibration buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0b) low conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0c) high conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0d) low conductivity Tris+high conductivity Histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0 Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0

HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 3091.2 n.d. 85.5 b 227 1 n.d. 77 c 26 0.2 n.d. 70.9 d 42 0.5 n.d. 83.8

EXAMPLE 5 Purification of a Bispecific Anti-Ang2/VEGF-A Antibody (IgG1Isotype) in a Protein A Chromatography

General conditions were according to the conditions described in Example2.

Antibody: anti-Ang2/VEGF-A

Initial load of PLBL2 determined in solution containing a bispecificanti-Ang2/VEGF-A antibody: 919.7 ng PLBL2/mg of antibody. Initial loadof CHOP determined in solution containing an anti-Ang2/VEGF-A: 682304 ngCHOP/mg of antibody.

Specific buffer conditions for Protein A chromatography

a) Control (wash with equilibration buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0b) low conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 25 mM Tris,25 mM NaCl, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0c) high conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0d) low conductivity Tris+high conductivity Histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0 Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0

HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 30351.0 n.d. 85.0 b 1707 0.8 n.d. 79.8 c 655 0.7 n.d. 52 d 1050 0.8 n.d.92.3

EXAMPLE 6 Purification of a Bispecific Anti-FIXa/X Antibody (IgG4Isotype) in a Protein A Chromatography

Purification of anti-FIXa/X antibody was tested in two differentchromatography settings:

Setting 1

General conditions were according to the conditions described in Example2.

Antibody: anti-FIXa/X

Initial load of PLBL2 determined in solution containing an anti-FIXa/Xantibody: 557 ng PLBL2/mg of antibody. Initial load of CHOP determinedin solution containing an anti-FIXa/X: 387377 ng CHOP/mg of antibody.

Specific buffer conditions for Protein A chromatography

a) high conductivity wash (with Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 700 mM Tris,pH 7.2 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 6: Wash IV:

Step 7: Elution: 50 mM acetic acid, pH 4.0b) low conductivity Tris+high conductivity Histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 2: Load Step3: Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0 Step 4: Wash II: 200 mMHis/1000 mM Tris, pH 7.0 Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH7.0 Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4.0

HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 163219.1 n.d. 79 b 2148 1.1 n.d. 77

Setting 2 General Chromatography Conditions

Column resin: Protein A material “Mab Select SuRe” (GE-Healthcare) Ø1cm, Height: 20.1 cm, CV: 15.79 ml

Equipment: Äkta Avant 150

Flow rate: 300 cm/h during all steps

A solution containing an anti-FIXa/X antibody, was applied to a ProteinA affinity column after equilibration (step 1) of the column.

Initial load of PLBL2 determined in solution containing an anti-FIXa/Xantibody: 557 ng PLBL2/mg of antibody.

The chromatographic steps were performed according to the followinggeneral scheme:

Step 1: Equilibration:

Step 2: Load of antibody containing solution

Step 3: Wash I Step 4: Wash II

Step 5: Wash III (additional wash)

Step 6: Elution

Specific buffer conditions for Protein A chromatography

a) high conductivity wash (with NaSO4 buffer only)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III:

Step 6: Elution: 35 mM acetic acid, pH 4.0b) low conductivity wash (Tris 1 mM)+high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 1 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4.0c) low conductivity wash (Tris 2 mM)+high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 35 mM acetic acid, pH 4.0d) low conductivity wash (Tris 4 mM)+high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 4 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4.0e) low conductivity wash (Tris 6 mM)+high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 6 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4.0f) low conductivity wash (Tris 4 mM, pH 7.8)+high conductivity wash(with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 4 mM Tris, pH 7.8

Step 6: Elution: 50 mM acetic acid, pH 4.0g) low conductivity wash (Tris 4 mM, pH 8.2)+high conductivity wash(with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:450 mM NaSO4, 20 mM NaAc, pH 4.8 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 4 mM Tris, pH 8.2

Step 6: Elution: 50 mM acetic acid, pH 4.0h) low conductivity wash (Tris 2 mM)+high conductivity wash (withHistidine (His)/Tris 1 M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:200 mM His/1000 mM Tris, pH 7.0 Step 4: Wash II: 20 mM NaPO4, pH 7.5Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 35 mM acetic acid, pH 4.0i) low conductivity wash (Tris 2 mM)+high conductivity wash (Histidine(His)/Tris 0.85 M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:200 mM His/850 mM Tris, pH 7.0 Step 4: Wash II: 20 mM NaPO4, pH 7.5 Step5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4.0j) low conductivity wash (Tris 2 mM)+high conductivity wash (Histidine(His)/Tris 0.7 M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:200 mM His/700 mM Tris, pH 7.0 Step 4: Wash II: 20 mM NaPO4, pH 7.5 Step5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4.0k) low conductivity wash (Tris 2 mM)+high conductivity wash (Histidine(His)/Tris 0.55 M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5 Step 2: Load Step 3: Wash I:200 mM His/550 mM Tris, pH 7.0 Step 4: Wash II: 20 mM NaPO4, pH 7.5 Step5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4.0

HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 1518204.2 n.d. 82 b 646 1 n.d. 73.8 c 737 1.2 n.d. 79 d 595 1.4 n.d. 78.5 e685 1.8 n.d. 79.5 f 692 1.4 n.d. 78.2 g 707 1.1 n.d. 76.4 h 299 0.5 n.d.79 i 140 0.4 n.d. 70 j 100 0.5 n.d. 71.9 k 112 0.7 n.d. 73

EXAMPLE 7 General Procedure/Conditions: Mock Cell Culture Fluid

Null harvested cell culture fluid was produced using non-transfectedCHO-DP12 cells cultured in serum-free media. Fermentation was performedat the 2 L-scale using a representative cell culture process. At the endof 14 days of fermentation, cell culture fluid was harvested viacentrifugation and sterile filtration. This harvested cell culture fluid(HCCF) was then stored at −70° C. until experimentation.

Purified PLBL2

Recombinant CHO PLBL2 with a C-terminal hexahistidine-tag was expressedin 35 L-scale transient transfections and purified from harvested cellculture fluid as previously described (Vanderlaan et al., 2015).Purified PLBL2 was then formulated in a PBS solution and stored at −70°C. until experimentation.

Purified Antibody

Recombinant humanized antibodies were expressed in CHO cells andpurified using column chromatography to ensure PLBL2 concentration wasbelow 20 ng/mg. Prior to beginning each study, each antibody wasbuffer-exchanged into PBS using PD-10 desalting columns (GE Healthcare).

Preparation of Load Material for Protein A Chromatography

To normalize the population and abundance of host cell proteins in theProtein A load across antibodies, purified antibodies were diluted tothe same concentration with PBS and spiked into HCCF from anon-producing cell line to give a final antibody titer of 5 g/L. Acontrol was also prepared wherein PBS was added instead of the purifiedantibody to evaluate non-specific host cell protein binding to theProtein A resin in the absence of antibody.

Packed-Bed Column Chromatography

All packed-bed column chromatography experiments were performed using a0.66 cm inner diameter by 20 cm bed height MabSelect SuRe (GEHealthcare) Protein A resin column. For each purification, the columnwas first equilibrated for 3 column volumes (CVs) with 25 mM tris, 25 mMNaCl, pH 7.7 (equilibration buffer). Then Protein A load was applied toa target load density of 30 g antibody/L resin, after which the columnwas washed for 3 CVs with equilibration buffer, 3 CVs of different typesof washing buffers, and again with 3 CVs of equilibration buffer.Subsequently, antibody was eluted at low pH with 0.1 to 0.15 M aceticacid, and eluate pools were collected starting at 0.5 OD at thebeginning of the elution peak; pooling was terminated after 2.8 CVs. Forthe control run with PBS-spiked null HCCF, a 2.8 CV mock elution poolwas generated starting from 1 CV to 3.8 CVs after the start of theelution phase. At the end of every run, each Protein A eluate was thentitrated to pH 5.0 using 1.5 M tris base. The column was then cleanedwith a 0.1 M sodium hydroxide solution. All phases had a volumetric flowrate of 20 CV/h except for the load, first equilibration wash, andelution phases, which had a flow rate of 15 CV/h.

-   -   A) Purification of an exemplary antibody (IgG4 isotype),        Antibody A, in a protein A chromatography

Specific washing buffer conditions for purification of Antibody A (IgG4isotype) using the general procedure of Example 7 (as outlined above):

-   -   a) 0.4 M Potassium Phosphate, pH 7.0    -   b) 25 mM Tris, 25 mM NaCl, pH 7.7    -   c) 0.75 M Arg—HCl, pH 7.0    -   d) 0.6 M NaCl, pH 7.0    -   e) Deionized water

Results:

HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817 Load: 7668 a 1434 167 b1440 107 c 331 40 d 1864 66 e 1204 31

-   -   B) Purification of an exemplary antibody (IgG1 isotype),        Antibody B, in a protein A chromatography

Specific washing buffer conditions for purification of Antibody B (IgG1isotype) using the general procedure of Example 7:

-   -   a) Deionized water

Results:

HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817 Load: 7668 a 2630 71

-   -   C) Purification of an exemplary antibody (IgG4 isotype),        Antibody C, in a protein A chromatography

Specific washing buffer conditions for purification of Antibody C (IgG4isotype) using the general procedure of Example 7:

-   -   a) 0.4 M Potassium Phosphate, pH 7.0    -   b) 25 mM Tris, 25 mM NaCl, pH 7.7    -   c) 0.75 M Arg—HCl, pH 7.0    -   d) 0.6 M NaCl, pH 7.0    -   e) Deionized water

Results:

HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817 Load: 7668 a 314 434 b 361369 c 214 82 d 780 214 e 410 68

-   -   D) Purification of an exemplary antibody (IgG1 isotype),        Antibody D, in a protein A chromatography

Specific washing buffer conditions for purification of Antibody D (IgG1isotype) using the general procedure of Example 7:

-   -   a) Deionized water

HCP total PLBL2 [ng/mg] [ng/mg] Load: 1067817 Load: 7668 a 6427 28

-   -   E) Purification of an exemplary antibody (IgG1 isotype),        Antibody E, in a protein A chromatography

Specific washing buffer conditions for purification of Antibody E (IgG1isotype) using the general procedure of Example 7:

-   -   a) 0.4 M Potassium Phosphate, pH 7.0    -   b) 31 mM Tris, pH 8.5    -   c) 55 mM Tris, pH 9.0    -   d) Deionized water

Results:

HCP total PLBL2 [ng/mg] [ng/mg] Load: 169706 Load: 333 a 1307 36 b 884 7c 601 0.2 d 929 8

-   -   F) Purification of an exemplary antibody (IgG1 isotype),        Antibody F, in a protein A chromatography

Specific washing buffer conditions for purification of Antibody F usingthe general procedure of Example 7:

-   -   a) 25 mM Tris, pH 9.0

Results:

HCP total PLBL2 [ng/mg] [ng/mg] Load: 994582 Load: 1363 a 806 0.5

1. Use of a low conductivity aqueous solution in a wash step of aprotein A chromatography for reducing the content of a host cell proteinwherein the protein A chromatography is used to purify a human IgG4 orIgG1 isotype antibody, wherein the low conductivity aqueous solution hasa conductivity value of about 0.5 mS/cm or less.
 2. Use according toclaim 1, wherein the host cell protein is phospholipase B-like 2 (PLBL2)or Clusterin.
 3. Use according to any one of claims 1 to 2, wherein thelow conductivity aqueous solution comprises about 0.1 mM to about 8 mMTris.
 4. Use according to any one of claims 1 to 2, wherein the lowconductivity aqueous solution comprises about 0.05 mM to about 2 mMpotassium phosphate.
 5. Use according any one of claims 1 to 4, whereinthe low conductivity aqueous solution has a pH of about 7 or higher. 6.Use according to any one of claims 1 to 5, wherein the low conductivityaqueous solution wash step is preceded or succeeded by a highconductivity aqueous solution wash step.
 7. Use according to claim 6,wherein the high conductivity aqueous solution has a conductivity valueof about 20 mS/cm or higher.
 8. Use according to any one of claims 6 to7, wherein the high conductivity aqueous solution comprises Histidine.9. Use according to any one of claims 1 to 8, wherein the human IgG4isotype antibody is an antibody against P-selectin or an antibodyagainst factor IXa and factor X or an antibody against IL-13 or anantibody against amyloid beta.
 10. Use according to any one of claims 1to 8, wherein the human IgG1 isotype antibody is an antibody againstInfluenza B or an antibody against VEGF-A or an antibody against CD22 ora bispecific antibody against HER3 and EGFR or an antibody againstamyloid beta or an antibody against Her2 or a bispecific antibodyagainst Ang2 and VEGF-A or a bispecific antibody againstcarcinoembryonic antigen (CEA) and CD3.
 11. Method for producing a humanIgG4 or IgG1 isotype antibody comprising the steps of a) cultivating acell comprising a nucleic acid encoding a human IgG4 or IgG1 isotypeantibody, b) recovering the human IgG4 or IgG1 isotype antibody from thecell or the cultivation medium, c) contacting the human IgG4 or IgG1isotype antibody with a protein A chromatography material, d) washingthe protein A chromatography material with a low conductivity aqueoussolution, wherein the low conductivity aqueous solution has aconductivity value of about 0.5 mS/cm or less, e) recovering the humanIgG4 or IgG1 isotype antibody from the protein A chromatography materialand thereby producing the human IgG4 or IgG1 isotype antibody. 12.Method for purifying a human IgG4 or IgG1 isotype antibody from a samplecomprising the steps of a) providing a sample comprising a human IgG4 orIgG1 isotype antibody, b) purifying the human IgG4 or IgG1 isotypeantibody with a protein A chromatography method/step, comprising washingthe protein A chromatography material with a low conductivity aqueoussolution, wherein the low conductivity aqueous solution has aconductivity value of about 0.5 mS/cm or less.
 13. Method according toany one of claims 11 to 12, wherein the amount of a host cell protein isreduced and wherein said host cell protein is phospholipase B-like 2(PLBL2) or Clusterin.
 14. Method according to any one of claims 11 to13, wherein the low conductivity aqueous solution comprises about 0.1 mMto about 8 mM Tris.
 15. Method according to any one of claims 11 to 14,wherein the low conductivity aqueous solution comprises about 0.05 mM toabout 2 mM potassium phosphate.
 16. Method according to any one ofclaims 11 to 15, wherein the low conductivity aqueous solution has a pHof about 7 or higher.
 17. Method according to any one of claims 11 to16, wherein the method additionally comprises washing the affinitychromatography material with a high conductivity aqueous solution and/orwith a medium conductivity aqueous solution before or after washing theprotein A chromatography material with low conductivity aqueoussolution.
 18. Method according to claim 17, wherein the highconductivity aqueous solution has a conductivity value of about 20 mS/cmor higher.
 19. Method according to claim 17, wherein the mediumconductivity aqueous solution has a conductivity value of from more than0.5 mS/cm to less than 20 mS/cm.
 20. Method according to any one ofclaims 17 to 19, wherein the high or medium conductivity aqueoussolution comprises Histidine.
 21. Method according to any one of claims11 to 20, wherein the human IgG4 isotype antibody is an antibody againstP-selectin or an antibody against factor IXa and factor X or an antibodyagainst IL-13 or an antibody against amyloid beta.
 22. Method accordingto any one of claims 11 to 20, wherein the human IgG1 isotype antibodyis an antibody against Influenza B or an antibody against VEGF-A or anantibody against CD22 or a bispecific antibody against HER3 and EGFR oran antibody against amyloid beta or an antibody against Her2 or abispecific antibody against Ang2 and VEGF-A or a bispecific antibodyagainst carcinoembryonic antigen (CEA) and CD3.